The testing of blood for various antigens and antibodies has become commonplace. For example, before a patient receives a blood transfusion, a number of laboratory tests must be performed on a sample of both the patient's and the donor's blood. Specifically, the blood must be typed, to determine its ABO and Rh D bloodgroup. Before a transfusion can proceed, the patient's serum must be tested for compatibility, or cross-matched, to determine whether the serum contains antibodies to antigens present on the erythrocytes (red blood cells) of a specific donor.
Additionally, there are an increasing number of antibodies that have been determined to be present in blood serum. Determining the presence or absence of certain antibodies can have great importance, both in the diagnosis and the treatment of certain disorders.
Blood typing, compatibility testing, and antibody screening are usually done through some type of agglutination immunoassay. Agglutination assays, due to their simplicity and wide range of applications, have become one of the most common methods used in blood testing. Agglutination assays can be performed relatively simply and can be detected or read visually without resort to expensive detection equipment. In a classic simple direct agglutination assay, e.,g agglutination of group A erythrocytes by anti-A sera, cells bearing a group A antigen are agglutinated directly by antibody.
One serious drawback to this type of testing is the tendency for erythrocytes possessing weak expression of a particular bloodgroup antigen being tested for not to agglutinate or clump to an extent so that proper visual determination of the positive response is possible. Moreover, in some cases, IgG class antibodies bind to the erythrocytes with high affinity but fail to induce agglutination. Addition of anti-IgG antibodies cross links the antibodies located on the erythrocytes and causes agglutination. An example of this type of reaction is the classic indirect Coombs test in which a test serum is first incubated with test erythrocytes, the cells are washed to remove excess unbound antibodies, and then mixed with an antiglobulin serum (Coombs reagent).
Lapierre et al. reported a variation of this agglutination assay that uses inert particles in the presence of a reagent that can be either an antibody or an antigen, for typing blood components that can be either erythrocytes or serum. Transfusion 30:109-113 (1990). For an indirect test, the reagent (e.g., serum) and blood components (e.g., erythrocytes) are mixed and incubated for a specified period of time. The mixture is centrifuged for about 10 minutes at 70.times.g into a bed of inert particles (i.e., a dextran gel) in a medium containing an agglutinating reagent in solution, generally an anti-human IgG (Coombs reagent). The erythrocytes that agglutinate are trapped within the matrix of inert particles, indicating a positive reaction. In a negative reaction, the erythrocytes circumvent the particles and pellet at the bottom of the reaction tube.
Because the Lapierre method indicates a positive response by trapping the agglutinated erythrocytes on the matrix of inert particles, weak positive reactions vary significantly in appearance. Generally, in a weak positive reaction, some of the erythrocytes will pellet in the bottom of the reaction tube and others will be trapped in multiple clumps. The size of the clumps of agglutinated erythrocytes will be dependent upon the strength of the antibody/antigen reaction. Thus, where the reaction is weak, the clumps will be smaller and more likely to pass through the particle matrix. Although the Lapierre method offers an easy-to-perform test, weak positive reactions may still be difficult to read.
Accordingly, there remains room in this field for improved techniques in blood typing assays, in making an assay that is easier, more flexible and more predictable to read where there is a weak positive response. The present invention provides an improved, flexible, rapid, and accurate bloodgrouping system. The result of such a test is fewer false negative responses.